The Pharmaceutical Industry has produced compounds of interest through fermentative production for a very long time. Generally, in a fermentation production facility, a compound (or compounds) of interest, such as certain organic compounds, proteins, carbohydrates, and the like can be produced in large quantities by culturing cells (often referred to as “host cell(s)”) in a liquid nutrient solution called medium. These host cells produce the compound(s) of interest either naturally or through genetic engineering or recombinant DNA technology.
In an embodiment, to culture the host cells, typically, the cells are submerged in a tank (often referred to as fermentor or bioreactor) of varying size containing a nutrient medium. This nutrient medium allows the cells to grow, multiply, and synthesize the compound(s) of interest. This process is often referred to as fermentation or cell culture. Harvesting the compound(s) of interest often requires extracting the compound(s) directly from the cells or from the supernatant.
Two commonly used fermentation systems are the continuous culture system and the fed-batch culture system, according to U.S. Pat. No. 5,639,658 to Drobish et al. The continuous culture system is typically used to extend the growth phase of the culture cells over long periods of time by providing fresh medium to the cells while simultaneously removing spent medium and cells from the fermentor. Such a culturing system serves to maintain optimal culturing conditions for certain host cell types and compounds of interest. The fed-batch fermentation system is generally defined as batch culture systems wherein fresh nutrients and/or other additives such as precursors to products are added as demanded by the fermentation process but no medium is withdrawn. There are three primary types of medium: chemically defined media, semi-defined media, and rich complex media.
A chemically defined medium as is illustrated in U.S. patent application Ser. No. 09/982,474, published on Apr. 4, 2002, is a medium essentially composed of chemically defined constituents. A semi-defined medium refers to a chemically defined medium supplemented with a small amount of complex nitrogen and/or carbon source(s), an amount as defined below, which typically is not sufficient to maintain growth of the micro-organism and/or the guarantee formation of a sufficient amount of biomass.
A rich complex medium is typically defined as a medium comprising a complex nitrogen and/or carbon source, such as soybean meal, cotton seed meal, corn steep liquor, yeast extract, casein hydrolysate, molasses, and the like. Likewise, a complex medium is a complete or nearly complete nutrient source for the microorganism. Rich complex media, in embodiments contain a carbon and a nitrogen source as well as vitamins, trace metals and minerals.
There are two primary types of fermentation: solid-state fermentation and aqueous fermentation. Solid-state fermentation includes the steps of cultivation of media, inoculation of the media with microorganisms, cultivation of the multi-organisms, extraction of biological products from the cultivated microorganisms and treatment of the waste materials from the culture. Solid-state fermentation is disclosed in U.S. Pat. No. 6,197,573 to Suryanarayan et al. Aqueous state fermentation is as disclosed below.
As is disclosed in U.S. Pat. No. 5,595,905 to Bishop et al, during a fermentation process, the bacteria or yeast growing in the fermentation broth consume nutrients at a variable rate. This rate is often related to such factors as the microorganism density and rate of growth. It is common in the fed-batch fermentation that the rate of consumption of nutrients will increase exponentially until an upper limit is reached for the fermentation that is often determined by the size of the fermentor or amount of nutrient and dissolved oxygen available in the medium.
As is disclosed in the prior art, it is desirable to maintain an adequate or sufficient concentration of nutrient in the medium. When the nutrient concentration is too high, either undesirable by-products, usually acetic acid, lactic acid or ethanol are produced, or growth inhibition is observed due to nutrient toxicity at higher concentrations[4]. When the nutrient concentration is too low the microorganism growth rate is restricted. Accordingly, the art field has strived to control the nutrient concentration in the medium. This is often involved different feeding patterns or measurements.
For example, in U.S. Pat. No. 5,595,905, patentees disclose taking samples from a culture medium, analyzing those samples, and based upon the analysis adding further nutrients to the water in an attempt to keep the nutrient concentration at constant in the culture. The '905 patent discloses using a computer to assist in monitoring the nutrient concentration.
U.S. Pat. No. 6,284,453 discloses general approaches to improving product formation that include, 1) using the best growth medium (carbon source, nitrogen source, precursors, and nutrients such as vitamins and minerals); 2) using the optimal pH, redox potential, agitation rate, aeration rate, ionic strength, osmotic pressure, water activity, hydrostatic pressure, and/or the like; 3) using the optimal dissolved oxygen or carbon dioxide concentration; 4) using inducers and repressors; 5) varying the above in a time-optimal fashion; 6) minimizing the accumulation of by-products that negatively impact the growth or metabolism of the organism, 7) genetically altering the organism using recombinant DNA or hybridoma technology; and, 8) using auxotrophic or mutants with altered regulatory systems, 9) and/or the like. Several methods have emerged to control growth and metabolism in a culture. As the '453 patent illustrates, various techniques use automated on-line or at-line measurements of the concentration of growth-limiting substrates such as glucose and glutamine. However, as can be imagined, feedback control based on substrate measurements can be relatively slow and less responsive. Likewise, another method that has met some success is to add growth-limiting substrate in an exponential manner. However, this exponential growth technique suffers from the drawbacks of allowing the culture medium to be underfed and/or overfed thereby not obtaining the optimal growth and metabolism characteristics. Another feedback measure of growth and metabolism is to measure the specific oxygen uptake rate and maintain it at a setpoint corresponding to the desired growth rate. This method is very effective in cultures with low growth rate. Another method is the dissolved oxygen-stat (DO-stat) method, which will be defined below. Likewise, another method includes pH-stat, which will also be defined below. Further methods include carbon dioxide transfer rate measurements, oxygen uptake measurements, respiratory quotient (RQ) measurements and the like. While these methods have proven successful under certain conditions, there are potential limitations associated with each method (see Table 1 for examples). Accordingly, the art field is in need of an improved measurement for monitoring and controlling the growth and metabolism characteristics of a culture.
The '453 patent discloses a novel method for controlling growth rate and metabolic state in a fed-batch fermentation by measuring the reagent addition rate, pH, oxygen uptake rate, biomass concentration, and reactor volume. The measurement of the reagent addition rate is divided by the measurement of the oxygen uptake rate and maintained at a pre-determined setpoint. Another embodiment is disclosed where a reagent addition rate is divided by the product of the biomass concentration and the reactor volume and maintained at a setpoint corresponding to a desired growth rate. A further additional embodiment is disclosed wherein the reagent addition rate and a specific oxygen uptake rate are maintained at different setpoints corresponding to a desired growth metabolic rate. However, as can be seen, the process disclosed on the '453 patent requires numerous measurements and calculations, and may be difficult to implement in commercial production on a routine basis. Therefore, the art field is in need of a process whereby simple measurements may be taken without complex calculations resulting in optimal growth and metabolic characteristics.
It is common for high cell density microbial fermentations to use a fed-batch mode of operation in order to resolve issues such as metabolic by-product accumulation or substrate inhibition, equipment limitation, etc. Cells are typically grown in batch mode to an intermediate cell density following which feeding of carbon/energy and/or complex nutrients is initiated. The feeding strategies can be classified into two major categories: (1) open-loop (non-feedback) and (2) closed-loop (feedback) feeding strategies.
The open-loop feeding strategies are typically pre-determined feed profiles for carbon/nutrient addition. There are an infinite number of feed profiles, but more commonly the feed rates are either constant or increased feed rate (either constant, stepwise or exponential) in order to keep up with the increasing cell densities. While these simple pre-determined feed profiles have been applied successfully in certain cases, the major drawback is the lack of feed rate adjustment based on metabolic feedback from the culture. Therefore, the open-loop feeding strategies can fail if an unexpected disturbance causes the culture to deviate from its “expected” growth pattern.
The closed-loop feeding strategies, on the other hand, rely on a measurement that is an indicator of the metabolic state of the culture. Two most commonly measured online variables, the dissolved oxygen (DO) concentration and pH, in microbial fermentation are also key indicators of cellular physiology. Therefore, they have traditionally been used as feedback variables upon which the feed rates are based. These more sophisticated closed-loop feeding strategies, called DO-stat and pH-stat which are based on the measurement of DO and pH respectively, have been utilized to minimize accumulation of inhibitory metabolites, such as acetate, during high cell density cultivation[1,2,3].
The traditional DO-stat control of nutrient feeding is simply based on the concept of DO rises (due to a reduction or cessation of oxygen consumption or respiration) upon nutrient limitation or depletion. The DO-stat control maintains the culture at a constant DO level (the DO setpoint) by increasing the nutrient feed rate when DO rises above the setpoint and reducing the nutrient feed rate when DO drops below the setpoint. The DO-stat strategy typically works well in defined media where nutrient depletion results in rapid DO rise. However, the DO-stat method often fails in media supplemented with rich complex nutrients such as yeast extract, tryptone, peptone, casamino acid, or Hy-Soy. Rich complex nutrients are capable of supporting cellular maintenance and respiration through amino acid catabolism such that the DO level remains low (i.e. no apparent DO spikes) even under carbon source limitation or depletion.
When a complex medium is used for culture growth, a pH-stat strategy may be more suitable than DO-stat since the culture pH tends to increase once the carbon source is depleted. In a manner similar to DO-stat control, the pH-stat method maintains a constant culture pH at about the setpoint by increasing the nutrient feed rate as pH rises above the setpoint and reducing the nutrient feed rate when pH drops below the setpoint. However, since the change in culture pH upon nutrient depletion is less responsive than that of DO, feeding control by pH-stat can be relatively sluggish when compared to DO-stat. In addition, the pH-stat control does not work well for culture grown in chemically defined media[3,4].
As explained before, in general, when the carbon source becomes limiting or depleted in fermentations employing complex medium, the culture pH rises while the DO value remains low. This suggests an active respiration in the absence of primary carbon source. This pH rise upon carbon source depletion is due to a combination of metabolism shift (to utilizing complex nitrogen which releases hydroxide after ammonium uptake) as well as reutilization of excreted acids (such as acetic acid). Similarly, the low DO value during carbon source depletion is most likely due to the metabolism shift to utilizing amino acids from the complex nitrogen feed. The degradation products of amino acids enter the tricarboxylic acid (TCA) cycle (e.g., 2-oxoglutarate, a deaminated product of glutamate, is an intermediate of the TCA cycle) and maintain active aerobic respiration, which results in oxygen consumption and low DO profile even under glucose depletion. Clearly the DO-stat control will not function properly if the culture DO concentration remains low during glucose limitation.
Therefore, the art field is in search of an improved method that can take advantage of the benefits offered by both pH and DO-stat without the usual drawbacks.